Upregulation of lncRNA LANCL1-AS1 inhibits the progression of non-small-cell lung cancer via the miR-3680-3p/GMFG axis

Abstract Patients with non-small-cell lung cancer (NSCLC) have a low survival rate. Long non-coding RNA (LncRNA) LANCL1 antisense RNA 1 (LANCL1-AS1) was indicated to be downregulated in NSCLC; however, its detailed function in NSCLC is unanswered. Real-time quantitative polymerase chain reaction revealed the downregulation of LANCL1-AS1 in NSCLC cell lines and subcellular fractionation assay showed that LANCL1-AS1 was mainly located in the cytoplasm of NSCLC cells. Cell counting kit-8, Transwell, and tube formation assays displayed that overexpression of LANCL1-AS1 suppressed NSCLC cell proliferation, migration, invasiveness, and angiogenesis in vitro. Animal experiments validated the tumor-suppressive role of LANCL1-AS1 in tumor-bearing mice. Mechanistically, LANCL1-AS1 upregulated glia maturation factor gamma (GMFG) expression by competitively binding to miR-3680-3p. GMFG knockdown reversed LANCL1-AS1 overexpression-mediated inhibitory impact on NSCLC malignant behaviors. Collectively, LANCL1-AS1 upregulation inhibits the progression of NSCLC by modulating the miR-3680-3p/GMFG axis.


Introduction
Lung cancer (LC) is the second most prevalent malignancy and the major cause of cancer-related deaths [1]. It is estimated in 2022 that LC accounts for 21% of all cancer-related deaths in men and women in the United States [2]. Emerging evidence has indicated that smoking is the main predisposing factor of LC [3]. Small-cell lung cancer (SCLC) and non-small-cell lung cancer (NSCLC) are two major types of LC, and NSCLC mainly incudes lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC), accounting for more than 80% of all LC cases [4]. Despite the declines in the incidence and mortality of LC owing to therapeutic advances and cessation, the 5-year relative survival rate of LC patients is 21%, while that of patients with metastatic disease is only 6% [5]. Hence, finding a more effective biomarker for the diagnosis and therapy of LC is urgent.
Long non-coding RNAs (lncRNAs) are RNA segments consisting of over 200 nucleotides which have no potential to encode proteins but have critical functions in various cellular processes by interacting with downstream molecules [6]. Plentiful studies have verified the involvement of lncRNAs in the tumorigenesis, pathogenesis, and angiogenesis of cancers, including NSCLC [7,8]. For example, LINC01599 is considered as an oncogene in LUAD by promoting autophagy [1]. LINC01296 knockdown inhibits the progression of NSCLC via the miR-143-3p/ATG2B [9]. Furthermore, the regulatory functions of lncRNAs are determined differently by their subcellular location. Numerous studies demonstrated that in the cytoplasm, lncRNAs work as competing endogenous RNAs (ceRNAs) to regulate downstream RNA expression [10]. LANCL1 antisense RNA 1 (LANCL1-AS1) is a novel lncRNA which was reported to be downregulated in NSCLC [11]. Additionally, LANCL1-AS1 was indicated to be an autophagy-related RNA in LUAD [1]. Bioinformatics analysis elucidated the downregulation of LANCL1-AS1 in lung tumors compared with adjacent normal tissues, indicating that LANCL1-AS1 might be a tumor suppressor of LC. In addition, lncLocator (http://www.csbio.sjtu.edu.cn/bioinf/lncLocator/) predicted that LANCL1-AS1 is largely distributed in the cytoplasm. Nevertheless, the detailed function of LANCL1-AS1 in NSCLC is unanswered.
This study aimed to probe the role as well as the regulatory mechanism of LANCL1-AS1 in NSCLC. We hypothesized that LANCL1-AS1 might act as a ceRNA to affect the pathogenesis of NSCLC by modulating downstream molecule expression. Our findings might help to develop a new idea for the diagnosis and therapy of NSCLC.

Transwell assay
Transwell assays were implemented for measuring cell migratory or invasive capabilities. NSCLC cells (2 × 10 4 ) were added to the upper chamber of the Transwell chamber (8 μm pore size; Corning, Lowell, MA, USA). The lower or upper chamber was added with complete medium or serum-free medium, respectively. After 48 h of incubation, the non-migratory cells in the upper chamber were swabbed and the migratory cells were subjected to 0.1% crystal violet staining. The invasion assay was similar except that the upper chamber was precoated with Matrigel (BD Biosciences, San Jose, CA, USA). Stained cells were imaged under a microscope (Nikon, Tokyo, Japan).

Tube formation assay
Human umbilical vein endothelial cell line (HUVEC) was obtained from American Type Culture Collection (ATCC, Manassas, VA, USA) and incubated in Endothelial Cell Growth Medium BulletKit (EGM, Lonza, Switzerland) at 37°C with 5% CO 2 in a humidified atmosphere. HUVECs (2 × 10 4 ) in 200 μL conditional medium from A549 and H460 cells were inoculated into a 24-well plate which was precoated with Matrigel (Corning) followed by incubation with 5% CO 2 at 37°C for 6 h. Tube structures were photographed under a bright-field microscope (Nikon). The mesh and length of the completed tubes were measured to quantify tube formation using Image View 3.7 (Jingtong, China).

In vivo xenograft experiments
BALB/c nude mice (male, 5-6 weeks) were obtained from Vital River (Beijing, China) and randomly divided into two groups (n = 5 per group). Recombinant lentivirus carrying LANCL1-AS1 (Lv-LANCL1-AS1) and empty control vector (Lv-con) were synthesized by GenePharma. Mice were injected subcutaneously with H460 cells (2 × 10 5 ) expressing Lv-LANCL1-AS1 or Lv-con. Tumor volume was monitored every 3 days and computed by the formula: volume = ½(length × width 2 ). On 18th day, the mice were sacrificed under anesthesia and tumor weight was measured. All animal experiments implemented were approved by the Ethics Committee of Gansu Provincial Hospital.

Statistical analysis
Data are expressed as the mean value ± standard deviation (SD). Statistical analysis was conducted using SPSS 21.0 (IBM Corp., Armonk, NY, USA). Student's t-test was utilized for two group comparison, while analysis of variance (ANOVA) was utilized for multiple group comparison followed by Tukey's post hoc analysis. p < 0.05 was regarded to be statistically significant.

LANCL1-AS1 is downregulated in NSCLC
LANCL1-AS1 expression was examined with GEPIA (http:// gepia.cancer-pku.cn/) which reveals LANCL1-AS1 downregulation in LUAD (Figure 1a). Kaplan-Meier Plotter (https://kmplot.com/analysis/) indicates that LANCL1-AS1 downregulation is associated with the poor prognosis of patients with LUAD (Figure 1b). Consistent with the above results, RT-qPCR disclosed a decreased level of LANCL1-AS1 in NSCLC cell lines (A549, H1299, and H460) in comparison to the normal cell line (HBE) (Figure 1c). In accord with the prediction of lncLocator in Figure 1d, the results of subcellular fractionation demonstrated that LANCL1-AS1 was largely located in the cytoplasm of NSCLC cells (Figure 1e). These results showed that a low level of LANCL1-AS1 is related to a poor prognosis of NSCLC.

GMFG is a target of miR-3680-3p
To further reveal the regulatory mechanism of LANCL1-AS1 in NSCLC, TargetScan was used for prediction of the downstream gene of miR-3680-3p. Three candidate mRNAs were screened out with the condition of cumulative weighted context++ score >0.7 (Figure 4a). RT-qPCR revealed the downregulation of miR-3680-3p in A549 and H460 cells treated with miR-3680-3p inhibitor ( Figure  4b). Additionally, only GMFG was upregulated in miR-3680-3p-depleted A549 and H460 cells (Figure 4c). Western blotting disclosed that depletion of miR-3680-3p increased the protein level of GMFG in A549 and H460 cells (Figure 4d and e). Then, we overexpressed miR-3680-3p in NSCLC cells using miR-3680-3p mimics ( Figure 4e). As shown in Figure 4f, upregulation of LANCL1-AS1 elevated GMFG protein expression in NSCLC cells, while overexpression of miR-3680-3p reversed the effect of LANCL1-AS1 upregulation. TargetScan predicts the existence of miR-3680-3p complementary site on GMFG 3'UTR and luciferase reporter assay further elucidated the binding relation between miR-3680-3p and GMFG (Figure 4g and h). GEPIA database shows the downregulation of GMFG in LUAD tissues compared with the normal samples (Figure 4i). In comparison to that in the normal cells, GMFG expression in NSCLC cells was markedly decreased (Figure 4j). Moreover, Kaplan-Meier Plotter exhibits a strong correlation between GMFG low expression and poor prognoses of LUAD patients (Figure 4k). Hence, GMFG is targeted by miR-3680-3p, and its expression is closely related to patient prognoses.

Overexpression of LANCL1-AS1 inhibits tumorigenesis of NSCLC in vivo
To further elucidate LANCL1-AS1 effect on NSCLC, in vivo experiments were carried out. Results displayed that the tumors in LANC1-AS1-overexpressed group grew much more slowly and smaller than those in the control group (Figure 6a-c). Notably, overexpression of LANCL1-AS1 markedly inhibited the expression of angiogenesis-associated proteins in tumors (Figure 6d). These indicated that LANCL1-AS1 exerts an inhibitory effect on tumorigenesis of NSCLC. Additionally, we detected the expression levels of LANCL1-AS1, miR-3680-3p, and GMFG in the tumors of each group. As depicted by the results, LANCL1-AS1 displayed a significantly higher level in Lv-LANCL1-AS1-treated group than that in the control group, confirming the successful overexpression of LNACL1-AS1 in the tumor xenograft mouse model (Figure 6e). Moreover, miR-3680-3p was downregulated and GMFG was markedly upregulated in LANCL1-AS1-overexpressed group (Figure 6f and g). These data indicated that LANCL1-AS1 upregulated GMFG expression by interacting with miR-3680-3p in vivo.

Discussion
In the present study, we found that lncRNA LANCL1-AS1 acted as a tumor suppressor in NSCLC by regulating the miR-3680-3p/GMFG axis. Our data showed that overexpression of LANCL1-AS1 inhibited NSCLC cell proliferation, migration, invasion as well as angiogenesis. This suppressive effect on the tumorigenesis of NSCLC was also verified in tumor-bearing mouse models. Despite the progress made in the diagnoses and treatments, the incidence of NSCLC is still high, with the overall 5-year survival rate lower than 17% [12]. Emerging evidence has illustrated the prominent effects of lncRNAs involved in regulation of cancers, including NSCLC. Multiple lncRNAs are considered as a prognostic biomarker and therapeutic target in NSCLC. For example, RP11-909N17.2 facilitates the cellular processes of NSCLC and predicts an adverse prognosis of patients [13]. PCAT29 inhibits NSCLC progression via the miR-494/PTEN axis and NSCLC patients with a higher level of PCAT29 have a better prognosis [14]. These suggest that lncRNAs can be a tumor suppressor gene or an oncogene. Here we probed the function of LANCL1-AS1 in NSCLC. LANCL1-AS1 is a novel gene that has been indicated to be downregulated in NSCLC; however, to our knowledge, the detailed functions of LANCL1-AS1 have not been studied yet. Currently, LANCL1-AS1 was shown to be downregulated in NSCLC cell lines and bioinformatics analysis revealed that its downregulation is associated with the adverse survival of LUAD patients, indicating the tumor suppressor role of LANCL1-AS1 in NSCLC. Moreover, gain-offunction assays demonstrated that LANCL1-AS1 overexpression restrained the proliferation, motion, and angiogenesis of NSCLC cells in vitro. Importantly, in vivo experiments displayed that upregulated LANC1-AS1 restrained tumorigenesis and angiogenesis in mice. These results elucidated that LANCL1-AS1 suppresses NSCLC progression.
Numerous studies have elucidated that in the cytoplasm, lncRNAs function as a ceRNA to competitively absorb microRNAs (miRNAs) and subsequently regulate messenger RNA (mRNA) expression [15]. For example, in prostate cancer, LINC01679 suppresses tumor progression by regulating miR-3150a-3p/SLC17A9 axis [16]. Additionally, in thyroid cancer, MIAT contributes to tumor progression by absorbing miR-150-5p and modulate EZH2 [17]. Bioinformatics analysis and our assays confirmed that LANCL1-AS1 was largely distributed in the cytoplasm of NSCLC cells, indicating the potential of LANCL1-AS1 as a ceRNA in NSCLC. With the use of bioinformatic tools and a series of experiments, we finally identified miR-3680-3p that could bind with LANCL1-AS1 in NSCLC cells. miR-3680-3p was shown to be sponged by circ-PRKCI to regulate AKT3 expression in esophageal squamous cell carcinoma [18]. Presently, it was shown that miR-3680-3p was upregulated in NSCLC (f) Western blotting for evaluating angiogenesis-related protein levels in NSCLC cells with above transfection. * p < 0.05, ** p < 0.01, *** p < 0.001. cells and its upregulation correlates with the adverse survival of LUAD patients.
It is recognized that miRNAs can bind to mRNA 3'UTRs by base-pairing, consequently leading to either mRNA degradation or translation suppression [19]. To better understand the mechanism of LANCL1-AS1, we screened the downstream gene of miR-3680-3p. Eventually, GMFG was singled out which displayed downregulated expression in NSCLC and strongly associated with the prognosis of LUAD patients. GMFG has been indicated to play a part in several cancers. For example, a high level of GMFG is related to adverse prognosis and facilitates the progression of epithelial ovarian cancer [20]. Moreover, GMFG exerts an antitumor role in breast cancer and is considered as a promising biomarker for the diagnosis and prognosis [21]. Intriguingly, GMFG was indicated to be downregulated in LUAD [22]. In this study, GMFG silencing was shown to reverse LANCL-AS1 overexpression-mediated inhibitory impact on NSCLC cell malignant behaviors, indicating the antitumor effect of the LANCL1-AS1/miR-3680-3p/GMFG axis in NSCLC.
In conclusion, we probed the role of LANCL1-AS1 in NSCLC. LANCL1-AS1 suppresses the migration, invasiveness, and angiogenesis of NSCLC cells and inhibits tumorigenesis in vivo. Mechanistically, LANCL1-AS1 exerts its antitumor effect by functioning as a ceRNA via the miR-3680-3p/GMFG axis. The findings might develop a novel idea for the therapy of NSCLC.